Adenylate cyclase (AC) activity in vessels is associated with vasodilation. The AC family includes 10 members of which AC9 is the least studied. We recently demonstrated that Adcy9 inactivation in mice protects from atherosclerosis in the aorta and potentiates endothelium-dependent vasodilation (EDV) to acetylcholine (ACh) in femoral arteries (FA). Our objectives were 1) to determine Adcy9 expression in FA; 2) investigate endothelial signalling pathways associated with Adcy9 inactivation-induced potentiation of ACh-induced EDV; and 3) to assess the effect of Adcy9 inactivation on EDV during flow-mediated dilation (FMD). FA were isolated from 8 to 13 week-old Adcy9- inactivated ( Adcy9 Gt ) and wild-type (WT) littermate mice. Adcy9 expression was studied in WT FA tissue section by in situ hybridization (ISH) using Adcy9 and negative control probes. Diameter of pressurized (80 mmHg) FA (internal diameter 288±3 μm) was studied in response to ACh (n=7-14) and shear stress (n=12) up to 20 dynes/cm 2 after preconstriction with phenylephrine. To study the role of endothelial signalling pathways in response to ACh-induced EDV, FA were incubated in absence or presence of inhibitors for nitric oxide (NO) synthase (L-NNA 0.1 μM), cyclooxygenase (meclofenamate 1 μM) and endothelial hyperpolarization (TRAM-34 10 μM plus apamin 0.1 μM). ISH with Adcy9 probe resulted in numerous dots in the vascular wall of the FA. Meclofenamate and the combination of TRAM-34 plus apamin decreased maximal (E max ) EDV to ACh, respectively, by 40 and 17% in Adcy9 Gt (P<0.01) but had no effect in WT mice. L-NNA reduced E max to ACh by 26% in WT (P<0.01) and 50% in Adcy9 Gt (P<0.01). Adcy9 inactivation tended to increase FMD to shear stress ranging from 6 to 20 dynes/cm 2 : At 10 dynes/cm 2 , FMD was increased from 25.4±4.9% in WT to 37.4±4.9% in Adcy9 Gt mice (P=0.083, n=12 per group). In conclusion, our data show that FA express Adcy9 and that Adcy9 inactivation increases the effects of endothelial NO, hyperpolarization and cyclooxygenase pathways in EDV. A numerical increase in FMD in Adcy9 Gt compared to WT confirms the potentiating effect of Adcy9 inactivation on EDV. The potentiation of endothelial dilatory pathways appears to be associated with the anti-atherosclerotic effects of Adcy9 inactivation.